Method of testing compounds for regulation of transcription of peroxisome proliferator activated receptor—gamma

ABSTRACT

In accordance with the present invention, there are provided a class of compounds which are capable of selectively modulating processes mediated by peroxisome proliferator activated receptor-gamma (PPAR-γ). The identification of such compounds makes possible the selective intervention in PPAR-γ mediated pathways, without exerting inadvertent effects on pathways mediated by other PPAR isoforms.

RELATED APPLICATIONS

This application is a 371 of PCT/US96/05465 filed Apr. 18, 1996 and adivisional of U.S. Ser. No. 08/465,375, filed Jun. 5, 1995, now issuedas U.S. Pat. No. 6,022,897, which is a continuation-in-part of U.S. Ser.No. 08/428,559, filed Apr. 25, 1995, now abandoned.

FIELD OF THE INVENTION

The present invention relates to methods for the modulation of nuclearreceptor mediated processes. In a particular aspect, the presentinvention relates to the use of a specific class of compounds for themodulation of processes mediated by peroxisome proliferator activatedreceptor-gamma (PPAR-γ). In another aspect, the present inventionrelates to methods of testing compounds for their ability to regulatetranscription-activating effects of PPAR-γ.

BACKGROUND OF THE INVENTION

Peroxisome proliferators are a structurally diverse group of compoundswhich, when administered to rodents, elicit dramatic increases in thesize and number of hepatic and renal peroxisomes, as well as concomitantincreases in the capacity of peroxisomes to metabolize fatty acids viaincreased expression of the enzymes required for the β-oxidation cycle(Lazarow and Fujiki, Ann. Rev. Cell Biol. 1:489-530 (1985); Vamecq andDraye, Essays Biochem. 24:1115-225 (1989); and Nelali et al., CancerRes. 48:5316-5324 (1988)). Chemicals included in this group are thefibrate class of hypolipidermic drugs, herbicides, and phthalateplasticizers (Reddy and Lalwani, Crit. Rev. Toxicol. 12:1-58 (1983)).Peroxisome proliferation can also be elicited by dietary orphysiological factors such as a high-fat diet and cold acclimatization.

Insight into the mechanism whereby peroxisome proliferators exert theirpieiotropic effects was provided by the identification of a member ofthe nuclear hormone receptor superfamily activated by these chemicals(Isseman and Green, Nature 347-645-650 (1990)). This receptor, termedperoxisome proliferator activated receptor alpha (PPARα), wassubsequently shown to be activated by a variety of medium and long-chainfatty acids and to stimulate expression of the genes encoding ratacyl-CoA oxidase and hydratase-dehydrogenase (enzymes required forperoxisomal β-oxidation), as well as rabbit cytochrome P450 4A6, a fattyacid ω-hydroxylase (Gottlicher et al., Proc. Natl. Acad. Sci. USA89:4653-4657 (1992); Tugwood et al., EMBO J. 11:433-439 (1992) Bardot etal., Biochem. Biophys. Res. Comm. 192:37-45 (1993); Muerhoff et al., J.Biol. Chem. 267:19051-19053 (1992); and Marcus et al., Proc. Natl. Acad.Sci. USA 90(12):5723-5727 (1993).

The above-noted references suggest a physiological role for PPARα in theregulation of lipid metabolism. PPARα activates transcription by bindingto DNA sequence elements, termed peroxisome proliferator responseelements (PPRE), as a heterodimer with the retinoid X receptor. Theretinoid X receptor is activated by 9-cis retinoic acid (see Kliewer etal., Nature 358:771-774 (1992), Gearing et al., Proc. Natl. Acad. Sci.USA 90:1440-1444 (1993), Keller et al., Proc. Natl. Acad. Sci. USA90:2160-2164 (1993), Heyman et al., Cell 68:397-406 (1992), and Levin etal., Nature 355:359-361 (1992)). Since the PPARα-RXR complex can beactivated by peroxisome proliferators and/or 9-cis retinoic acid, theretinoid and fatty acid signaling pathways are seen to converge inmodulating lipid metabolism.

Since the discovery of PPARα, additional isoforms of PPAR have beenidentified, e.g., PPARβ, PPARγand PPARδ, which are spatiallydifferentially expressed. Because there are several isoforms of PPAR, itwould be desirable to identify compounds which are capable ofselectively interacting with only one of the PPAR isoforms. Suchcompounds would find a wide variety of uses, such as, for example, inthe prevention of obesity, for the treatment of diabetes, and the like.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have identified a class ofcompounds which are capable of selectively modulating processes mediatedby peroxisome proliferator activated receptor-gamma (PPAR-γ). Theidentification of such compounds makes possible the selectiveintervention in PPAR-γ mediated pathways, without exerting inadvertenteffects on pathways mediated by other PPAR isoforms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the activation of a GAL4-PPARγ fusion protein by avariety of prostaglandin or prostaglandin-like compounds. In the figure,black bars represent 15-deoxy-Δ^(12,14)-prostaglandin-J₂ (15-d PGJ2),the dark, striped bars represent prostaglandin-J₂ (PGJ2), the darklyshaded bars represent 9α, 11β-prostaglandin-F₂ (9a, 11bPGF2), the light,closely (diagonally) striped bars represent prostaglandin-I₂ (PGI2), theopen bars represent prostaglandin-A₂ (PGA2), the dark bars with lightdots represent prostaglandin-B₂ (PGB2), the horizontally hatched barsrepresent prostaglandin-D₂ (PGD2), the light bars with dark dotsrepresent prostaglandin-E. (PGE2), the light, sparsely (diagonally)hatched bars represent prostaglandin-F_(2α) (PGF2a), and the light barswith sparsely spaced dots represent bicycloprostaglandin-E₁ (BicycloE1).

FIG. 2 illustrates the dose response for activation of a GAL4-PPARγfusion protein by a variety of prostaglandin or prostaglandin-likecompounds. In the figure, open circles represent prostaglandin-D₂(PGD2), darkened circles represent prostaglandin-J₂ (PGJ2), open squaresrepresent Δ¹²-prostaglandin-J₂ (Δ12-PGJ2), and darkened squaresrepresent 15-deoxy-Δ^(12,14)-prostaglandin-J₂ (15-deoxy-Δ12,14-PGJ2).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided methods formodulating process(es) mediated by peroxisome proliferator activatedreceptor-gamma (PPAR-γ), said method comprising conducting saidprocess(es) in the presence of at least one PPAR-γ-selectiveprostaglandin or prostaglandin-like compound or precursor thereof.

PPAR-γ-selective prostaglandins or prostaglandin-like compoundscontemplated for use in the practice of the present invention includemembers of the prostaglandin-J₂ family of compounds (e.g.,prostaglandin-J₂, Δ¹²-prostaglandin-J₂ or15-deoxy-Δ^(12,14)-prostaglandin-J₂), members of the prostaglandin-D₂family of compounds (e.g., prostaglandin-D₂), or precursors thereof, aswell as compounds having the structure I:

wherein:

A is selected from hydrogen or a leaving group at the α- or β-positionof the ring, or A is absent when there is a double bond between C^(α)and C^(β) of the ring;

X is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynylor substituted alkynyl group having in the range of 2 up to 15 carbonatoms; and

Y is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynylor substituted alkynyl group having in the range of 2 up to 15 carbonatoms.

As employed herein, the term “leaving group” refers to functional groupswhich can readily be removed from the precursor compound, for example,by nucleophilic displacement, under E₂ elimination conditions, and thelike. Examples include hydroxy groups, alkoxy groups, tosylates,brosylates, halogens, and the like.

As employed herein, “lower alkyl” refers to straight or branched chainalkyl groups having in the range of about 1 up to 4 carbon atoms;“alkyl” refers to straight or branched chain alkyl groups having in therange of about 1 up to 12 carbon atoms; “substituted alkyl” refers toalkyl groups further bearing one or more substituents such as hydroxy,alkoxy (of a lower alkyl group), mercapto (of a lower alkyl group),halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate,sulfonyl, sulfonamide, and the like.

As employed herein, “cycloalkyl” refers to cyclic ring-containing groupscontaining in the range of about 3 up to 8 carbon atoms, and“substituted cycloalkyl” refers to cycloalkyl groups further bearing oneor more substituents as set forth above.

As employed herein, “alkenyl” refers to straight or branched chainhydrocarbyl groups having at least one carbon-carbon double bond, andhaving in the range of about 2 up to 12 carbon atoms and “substitutedalkenyl” refers to alkenyl groups further bearing one or moresubstituents as set forth above.

As employed herein, “alkynyl” refers to straight or branched chainhydrocarbyl groups having at least one carbon-carbon triple bond, andhaving in the range of about 2 up to 12 carbon atoms, and “substitutedalkynyl” refers to alkynyl groups further bearing one or moresubstituents as set forth above.

As employed herein, “aryl” refers to aromatic groups having in the rangeof 6 up to 14 carbon atoms and “substituted aryl” refers to aryl groupsfurther bearing one or more substituents as set forth above.

As employed herein, “alkylaryl” refers to alkyl-substituted aryl groupsand “substituted alkylaryl” refers to alkylaryl groups further bearingone or more substituents as set forth above.

As employed herein, “arylalkyl” refers to aryl-substituted alkyl groupsand “substituted arylalkyl” refers to arylalkyl groups further bearingone or more substituents as set forth above.

As employed herein, “arylalkenyl” refers to aryl-substituted alkenylgroups and “substituted arylalkenyl” refers to arylalkenyl groupsfurther bearing one or more substituents as set forth above.

As employed herein, “arylalkynyl” refers to aryl-substituted alkynylgroups and “substituted arylalkynyl” refers to arylalkynyl groupsfurther bearing one or more substituents as set forth above.

As employed herein, “aroyl” refers to aryl-carbonyl species such asbenzoyl and “substituted aroyl” refers to aroyl groups further bearingone or more substituents as set forth above.

As employed herein, “heterocyclic” refers to cyclic (i.e.,ring-containing) groups containing one or more heteroatoms (e.g., N, O,S, or the like) as part of the ring structure, and having in the rangeof 3 up to 14 carbon atoms and “substituted heterocyclic” refers toheterocyclic groups further bearing one or more substituents as setforth above.

As employed herein, “acyl” refers to alkyl-carbonyl species.

As employed herein, “halogen” or “halo” refers to fluoro substituents,chloro substituents, bromo substituents or iodo substituents.

In a presently preferred aspect of the present invention, “X” of FormulaI is selected from:

—(CRR)_(m)—Z,

—(CRR)_(m′)—C (R)═C(R)—(CRR)_(m′)—Z, or

—(CRR)_(m″)—C≡C—(CRR)_(m″)—Z, wherein:

each R is independently selected from H, lower alkyl, substituted loweralkyl, hydroxy, lower alkoxy, thioalkyl, halogen, trifluoromethyl,cyano, nitro, amino, carboxyl, carbamate, sulfonyl or sulfonamide,

m falls in the range of 1 up to 15,

each m′ falls independently in the range of 0 up to 12, with the provisothat the total chain length of the alkenyl moiety does not exceed 15carbon atoms,

each m″ falls independently in the range of 0 up to 12, with the provisothat the total chain length of the alkynyl moiety does not exceed 15carbon atoms, and

Z is a polar, heteroatom-containing substituent.

Those of skill in the art can readily identify numerous groups whichsatisfy the requirement that Z be a polar, heteroatom-containing (i.e.,O, N, S, or the like) substituent. Thus, Z can be selected from cyano,nitro, amino, carbamate, or a substituent having the structure:

—CH₂OR′, wherein R′ is selected from H, alkyl, alkenyl, alkynyl, acyl,aryl, or the like;

—C(O)R″, wherein R″ is selected from H, alkyl, substituted alkyl,alkoxy, alkylamino, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, aryloxy, arylamino, alkylaryl,substituted alkylaryl, arylalkyl, substituted arylalkyl, heterocyclic,substituted heterocyclic or trifluoromethyl,

—CO₂R′″, wherein R′″ is selected from H, alkyl, alkenyl, alkynyl, or thelike;

—SR′, —S(O)R′, —S(O)₂R′ or —S(O)₂NHR′, wherein each R′ is as definedabove,

and the like.

Especially preferred compounds employed in the practice of the presentinvention are those wherein “X” of Formula I is

—CRR—C(R)═C(R)—(CRR)_(m)—Z, wherein:

each R is independently selected from H, lower alkyl, substituted loweralkyl, hydroxy, alkoxy (of a lower alkyl group), halogen,trifluoromethyl, amino, carboxyl or sulfonyl,

m falls in the range of 1 up to 6, and

z is selected from —CH₂OH, —CH₂OAc, —CO₂H, —CO₂Me or —CO₂Et.

In another preferred aspect of the present invention, “Y” of Formula Iis selected from:

═C(R)—[C(R)═C(R)]_(n)—(CRR)_(n′)—Z′ (II),

═C(R)—[C═C]_(n″)—(CRR)_(n′)—Z′ (IIA),

═C(R)—CRR—CR(R′)—(CRR)_(n′)—Z′ (III),

—[C(R)═C(R)]_(n′)—(CRR)_(n′)—Z′ (IV), or

—[C≡C]_(n)—(CRR)_(n′)—Z′ (IVA),

wherein

each R is independently as defined above,

each R′ is independently selected from H, lower alkyl, substituted loweralkyl or a leaving group,

Z′ is selected from H, lower alkyl or substituted lower alkyl,

n falls in the range of 0 up to 4,

n′ falls in the range of 2 up to 12, and

n″ falls in the range of 1 up to 3.

Especially preferred compounds contemplated for use in the practice ofthe present invention include those wherein “Y” of Formula I is selectedfrom:

═C(R)—C(R)═C(R)—(CRR)_(n′)—Z′ (II),

═C(R)—CRR—CR(R′)—(CRR)_(n′)—Z′ (III), or

—C(R)═C(R)—CR(R′)—(CRR)_(n′)—Z′ (IV), wherein

each R is independently as defined above,

each R′ is independently as defined above,

Z′ is selected from H, lower alkyl or substituted lower alkyl, and

n′ falls in the range of 1 up to 6.

Presently most preferred compounds for use in the practice of thepresent invention include those wherein “Y” of Formula I is

═C(R)—C(R)═C(R)—(CRR)_(n′)—Z′  (II),

wherein each R is selected from H, lower alkyl or substituted loweralkyl, n is 1, n′ falls in the range of about 2 up to 6, and Z′ isselected from H or lower alkyl; or those wherein “Y” of Formula I is

═C(R)—CRR—CR(R′)—(CRR)_(n′)—Z′  (III) or

—C(R)═C(R)—CR(R′)—(CRR)_(n′)Z′  (IV),

wherein each R is selected from H, lower alkyl or substituted loweralkyl, R′ is selected from H, lower alkyl, or an hydroxy group, n is 1,n′ falls in the range of about 2 up to 6, and Z′ is selected from H orlower alkyl.

Referring to the structural formulae set forth above, prostaglandin-D₂(Pg-D2) is described by Formula I (as set forth above), wherein A is9-OH, Y is IV, each R is hydrogen, R′ is hydroxy, Z is —CO₂H, m is 3, Z′is methyl, n is 1 and n′ is 4; prostaglandin-J₂ (Pg-J2) is described byFormula I, wherein A is absent, Y is IV, each R is hydrogen, R′ ishydroxy, Z is —CO₂H, m is 3, Z′ is methyl, n is 1 and n′ is 4;Δ¹²-prostaglandin-J₂ (Δ¹²-Pg-J2) is described by Formula I, wherein A isabsent, Y is III, each R is hydrogen, R′ is hydroxy, Z is —CO₂H, m is 3,Z′ is methyl, n is 1 and n′ is 4; 15-deoxy-Δ^(12,14)-prostaglandin-J₂(15-deoxy-Δ^(12,14)-Pg-J2) is described by Formula I, wherein A isabsent, Y is II, each R is hydrogen, Z is —CO₂H, m is 3, Z′ is methyl, nis 1 and n′ is 4.

The above-described compounds can be readily prepared using a variety ofsynthetic methods, as are well known by those of skill in the art. Forexample, many of the above-described compounds can be preparedchemically or enzymatically, from the naturally occurring precursor,arachidonic acid.

As employed herein, the term “modulate” refers to the ability of amodulator for a member of the steroid/thyroid superfamily to eitherdirectly (by binding to the receptor as a ligand) or indirectly (as aprecursor for a ligand or an inducer which promotes production of ligandfrom a precursor) induce expression of gene(s) maintained under hormoneexpression control, or to repress expression of gene(s) maintained undersuch control.

As employed herein, the phrase “processes mediated by PPARγ” refers tobiological, physiological, endocrinological, and other bodily processeswhich are mediated by receptor or receptor combinations which areresponsive to the PPAR-γ-selective prostaglandin or prostaglandin-likecompounds described herein. Such processes include cell differentiationto produce lipid-accumulating cells, modulation of blood glucose levelsand insulin sensitivity, regulation of leptin levels and subsequentfeeding levels (for the control of satiety and/or appetite), regulationof thermogenesis and fatty acid metabolism, regulation of fat levels forthe treatment of lipodystrophies, control of cell differentiation forthe treatment of myxoid liposarcomas, regulation of triglyceride levelsand lipoproteins for the treatment of hyperlipidemia, modulation ofgenes expressed in adipose cells (e.g., leptin, lipoprotein, lipase,uncoupling protein, and the like), and the like.

In accordance with the present invention, modulation of processesmediated by PPARγ can be accomplished in vitro or in vivo. In vivomodulation can be carried out in a wide range of subjects, such as, forexample, humans, rodents, sheep, pigs, cows, and the like.

PPAR-γ-selective prostaglandin or prostaglandin-like compoundscontemplated for use in the practice of the present invention can beemployed for both in vitro and in vivo applications. For in vivoapplications, the invention compounds can be incorporated into apharmaceutically acceptable formulation for administration. Those ofskill in the art can readily determine suitable dosage levels whencompounds contemplated for use in the practice of the present inventionare so used.

In accordance with another embodiment of the present invention, there isprovided a method of testing compound(s) for the ability to regulate thetranscription-activating effects of a peroxisome proliferator activatedreceptor-gamma. (PPAR-γ), said method comprising assaying for changes inthe level of reporter protein present as a result of contacting cellscontaining said receptor and reporter vector with said compound;

wherein said reporter vector comprises:

(a) a promoter that is operable in said cell,

(b) a hormone response element, and

(c) a DNA segment encoding a reporter protein,

wherein said reporter protein-encoding DNA segment is operatively linkedto said promoter for transcription of said DNA segment, and

wherein said hormone response element is operatively linked to saidpromoter for activation thereof.

Hormone response elements contemplated for use in the practice of thepresent invention are composed of at least one direct repeat of two ormore half sites separated by a spacer of one nucleotide. The spacernucleotide can be selected from any one of A, C, G or T. Each half siteof response elements contemplated for use in the practice of theinvention comprises the sequence

-RGBNNM-,

wherein

R is selected from A or G;

B is selected from G, C, or T;

each N is independently selected from A, T, C, or G; and

M is selected from A or C;

with the proviso that at least 4 nucleotides of said -RGBNNM-sequenceare identical with the nucleotides at corresponding positions of thesequence -AGGTCA-. Response elements employed in the practice of thepresent invention can optionally be preceded by N_(x), wherein x fallsin the range of 0 up to 5.

Presently preferred response elements contain at least one copy (withone, two or three copies most common) of the minimal sequence:

AGGACA A AGGTCA (SEQ ID NO:5).

As noted above, the minimal sequence can optionally be flanked byadditional residues, for example, as in the sequence:

GGACC AGGACA A AGGTCA CGTTC (SEQ ID NO:6).

In a preferred embodiment of the present invention, only the ligandbinding domain of PPARγ is utilized, in combination with the DNA bindingdomain of GAL4 protein, for the identification of PPARγ ligands orligand-precursors. This allows one to avoid possible background signalcaused by the potential presence of endogenous PPARγ in the host cellsused for the assay.

The DNA binding domain of the yeast GAL4 protein comprises at least thefirst 74 amino acids thereof (see, for example, Keegan et al., Science231:699-704 (1986)). Preferably, the first 90 or more amino acids of theGAL4 protein will be used, with the first 147 amino acid residues ofyeast GAL4 being presently most preferred.

The GAL4 fragment employed in the practice of the present invention canbe incorporated into any of a number of sites within the PPARγ receptorprotein. For example, the GAL4 DNA binding domain can be introduced atthe amino terminus of the PPARγ receptor protein, or the GAL4 DNAbinding domain can be substituted for the native DNA binding domain ofthe PPARγ receptor, or the GAL4 DNA binding domain can be introduced atthe carboxy terminus of the PPARγ receptor protein, or at otherpositions as can readily be determined by those of skill in the art.Thus, for example, a modified receptor protein can be prepared whichconsists essentially of amino acid residues 1-147 of GAL4, plus theligand binding domain of PPARγ (i.e., containing the ligand bindingdomain only of said receptor (i.e., residues 163-475 of SEQ ID NO:1),substantially absent the DNA binding domain and amino terminal domainthereof).

Identification methods according to the present invention involve theuse of a functional bioassay system, wherein the modified receptor and areporter plasmid are cultured in suitable host cells in the presence oftest compound. Evidence of transcription (e.g., expression) of reportergene is then monitored to determine the presence of an activatedreceptor-ligand complex. Accordingly, the functional bioassay systemutilizes two plasmids: an “expression” plasmid and a “reporter” plasmid.The expression plasmid can be any plasmid which contains and is capableof expressing DNA encoding the desired form of PPARγ receptor protein(i.e., intact receptor or GAL4 chimeric receptor as describedhereinabove), in a suitable host cell. The reporter plasmid can be anyplasmid which contains an operative PPRE or GAL4 response element, asappropriate, functionally linked to an operative reporter gene.

Exemplary PPREs have been described in detail hereinabove. ExemplaryGAL4 response elements am those containing the palindromic 17-mer:

5′-CGGAGGACTGTCCTCCG-3′ (SEQ ID NO:7).

such as, for example, 17MX, as described by Webster et al., in Cell52:169-178 (1988), as well as derivatives thereof. Additional examplesof suitable response element include those described by Hollenberg adEvans in Cell 55:899-906 (1988); or Webster at al. in Cell54:199-207(1988).

Exemplary reporter genes include chloramphenicol transferase (CAT),luciferase (LUC), beta-galactosidase (β-gal), and the like. Exemplarypromoters include the simian virus (SV) promoter or modified formthereof (e.g., ΔSV), the thymidine kinase (TK) promoter, the mammarytumor virus (MTV) promoter or modified form thereof (e.g., ΔMTV), andthe like [see, for example, Mangelsdorf et al., in Nature 345:224-229(1990), Mangelsdorf et al., in Cell 66:555-561 (1991), and Berger etal., in J. Steroid Biochem. Molec. Biol. 41:733-738 (1992)]. Theplasmids pGMCAT, pGHCAT, pTK-GAL_(p)3-LUC, ΔMTV-GAL_(p)3-LUC,ΔMTV-GAL_(p)3-CAT, and the like, are examples of reporter plasmids whichcontain an operative hormone responsive promoter/enhancer elementfunctionally linked to an operative reporter gene, and can therefore beused in the above-described functional bioassay (see Example 2 fordetails on the preparation of these plasmids). In pGMCAT, the operativehormone responsive promoter/enhancer element is the MTV LTR; in FLpGHCAT it is the functional portion of the growth hormone promoter. Inboth pGMCAT and GHCAT the operative reporter gene is the bacterial genefor chloramphenicol acetyltransferase (CAT).

As used herein in the phrase “operative response element functionallylinked to an operative reporter gene”, the word “operative” means thatthe respective DNA sequences (represented by the terms “PPRE,” “GAL4response element” and “reporter gene”) are operational, i.e., work fortheir intended purposes; the word “functionally” means that after thetwo segments are linked, upon appropriate activation by aligand-receptor complex, the reporter gene will be expressed as theresult of the fact that the “PPRE” or “GAL4 response element” was“turned on” or otherwise activated.

In practicing the above-described functional bioassay, the expressionplasmid and the reporter plasmid are co-transfected into suitable hostcells. The transfected host cells are then cultured in the presence andabsence of a test compound to determine if the test compound is able toproduce activation of the promoter operatively linked to the PPRE orGAL4 response element of the reporter plasmid. Thereafter, thetransfected and cultured host cells are monitored for induction (i.e.,the presence) of the product of the reporter gene sequence.

Any cell line can be used as a suitable “host” for the functionalbioassay contemplated for use in the practice of the present invention.Thus, in contrast to the requirements of prior art assay systems, whenGAL4 chimerics are employed, there is no need to use receptor-negativecells in carrying out the invention process. Since the modified receptoremployed in the practice of the present invention is the only species inthe test cell which is capable of initiating transcription from a GAL4response element, the expression of native receptor by the test celldoes not contribute to background levels. Thus, the invention bioassaycan be made to be very selective.

Cells contemplated for use in the practice of the present inventioninclude transformed cells, non-transformed cells, neoplastic cells,primary cultures of different cell types, and the like. Exemplary cellswhich can be employed in the practice of the present invention includeSchneider cells, CV-1 cells, HuTu80 cells, F9 cells, NTERA2 cells, NB4cells, HL-60 cells, 293 cells, Hela cells, yeast cells, and the like.Preferred host cells for use in the functional bioassay system are COScells and CV-1 cells. COS-1 (referred to as COS) cells are monkey kidneycells that express SV40 T antigen (Tag); while CV-1 cells do not expressSV40 Tag. The presence of Tag in the COS-1 derivative lines allows theintroduced expression plasmid to replicate and provides a relativeincrease in the amount of receptor produced during the assay period.CV-1 cells are presently preferred because they are particularlyconvenient for gene transfer studies and provide a sensitive andwell-described host cell system.

The above-described cells (or fractions thereof) are maintained underphysiological conditions when contacted with physiologically activecompound. “Physiological conditions” are readily understood by those ofskill in the art to comprise an isotonic, aqueous nutrient medium at atemperature of about 37° C.

In accordance with another embodiment of the present invention, there isprovided a method of screening for antagonists of PPARγ receptorproteins, said method comprising

culturing test cells containing

(i) increasing concentrations of at least one compound whose ability toinhibit the transcription activation activity of PPARγ agonists issought to be determined, and

(ii) optionally, at least one PPARγ agonist,

wherein said test cells contain

(i) exogenous DNA which expresses intact PPARγ or a modified form ofPPARγ, wherein the modified form of PPARγ contains the DNA bindingdomain of GAL4, and

(ii) a PPRE or GAL4 response element, respectively, operatively linkedto a reporter gene; and thereafter

assaying for evidence of transcription of said reporter gene in saidcells as a function of the concentration of said compound in saidculture medium, thereby indicating the ability of said compound toinhibit activation of transcription by PPARγ agonists.

Media employed for such culturing may include agonist for the receptorbeing tested, or the receptor may be constitutive (i.e., not require thepresence of agonist for activation), or a fixed concentration of agonistcan be added to the media employed for such testing.

The above-described assays of the present invention have low backgroundand a broad dynamic range.

In accordance with yet another embodiment of the present invention,there is provided a method for preventing obesity, said methodcomprising administering to a subject in need thereof an amount of aperoxisome proliferator activated receptor-gamma (PPAR-γ) antagonisteffective to block cell differentiation to produce lipid-accumulatingcells.

As employed here, “obesity” refers generally to individuals who are atleast about 20-30% over the average weight for his/her age, sex andheight. Technically, “obese” is defined, for males, as individuals whosebody mass index is greater than 27.8 kg/m², and for females, asindividuals whose body mass index is greater than 27.3 kg/m².

Those of skill in the art recognize that there are numerous cell typeswhich are capable of differentiation to produce “lipid-accumulatingcells,” such as, for example, mesenchymal cells (e.g., fibroblasts).

As employed herein, the phrase “amount . . . effective to block celldifferentiation” refers to levels of compound sufficient to providecirculating concentrations high enough to effect activation of PPARγ.Such a concentration typically falls in the range of about 10 nM up to 2μM; with concentrations in the range of about 100 nM up to 200 nM beingpreferred.

In accordance with a particular embodiment of the present invention,compositions comprising at least one prostaglandin or prostaglandin-likecompound (as described above), and a pharmaceutically acceptable carrierare contemplated. Exemplary pharmaceutically acceptable carriers includecarriers suitable for oral, intravenous, subcutaneous, intramuscular,intracutaneous, and the like administration. Administration in the formof creams, lotions, tablets, dispersible powders, granules, syrups,elixirs, sterile aqueous or non-aqueous solutions, suspensions oremulsions, and the like, is contemplated.

For the preparation of oral liquids, suitable carriers includeemulsions, solutions, suspensions, syrups, and the like, optionallycontaining additives such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents, and the like.

For the preparation of fluids for parenteral administration, suitablecarriers include sterile aqueous or non-aqueous solutions, suspensions,or emulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized, for example,by filtration through a bacteria-retaining filter, by incorporatingsterilizing agents into the compositions, by irradiating thecompositions, or by heating the compositions. They can also bemanufactured in the form of sterile water, or some other sterileinjectable medium immediately before use.

In accordance with still another embodiment of the present invention,there is provided a method for treating diabetes, said method comprisingadministering to a subject in need thereof an amount of a peroxisomeproliferator activated receptor-gamma (PPAR-γ) agonist effective tolower the blood glucose level of said subject.

As employed herein, the phrase “amount . . . effective to lower bloodglucose levels” refers to levels of compound sufficient to providecirculating concentrations high enough to accomplish the desired effect.Such a concentration typically falls in the range of about 10 nM up to 2μM; with concentrations in the range of about 100 nM up to 200 nM beingpreferred.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 Preparation of GAL4-receptor fusion Proteins

A basic vector usefull for the generation of GAL4-receptor fusionproteins is called pCMX-GAL4 (see SEQ ID NO:3). This vector encodes GAL4DNA binding do followed by a polylinker sequence useful in the cloning.The parental expression vector pCMX has been described by Umesono etal., in Cell 65:1255-1266 (1991), and the GAL4 portion of pCMX-GAL4 isderived from plaid pSG424, described by Sadowski ad Ptashne, in NucleicAcids Res. 11:7539 (1989).

In general, GAL4-receptor ligand binding domain fusions are prepared bytaking advantage of mutant receptor cDNA clones, such as GR-RAR chimera[see, for example, Giguere et al., in Nature 330:624-629 (1987)]. Thesemutant receptor cDNAs encode common XhoI sites at the end of the DNAbinding domain, as described by Giguere et al., supra. To do so, a newpCMX-GAL4 vector was prepared which encodes a compatible SalI site inthe polylinker sequence (there is an XhoI site in the GAL4 sequence):

SalI site: G′TCGAC

XhoI site: C′TCGAG

This allows efficient transfer of the receptor ligand binding domain toGAL4 DNA binding domain. Through this method, a number of chimericspecies have been generated, including GAL4-PPARγ, containing residues163-475 of PPARγ (see SEQ ID NO:1).

If mutants of the type referred to above are 25 not available for theconstruction of GAL4-containing chimerics, one may simply look for anyconvenient restriction enzyme site within or downstream of the DNAbinding domain of the receptor of interest (i.e., within about the first30 amino acid residues downstream of the conserved Gly-Met residues ofthe DNA binding domain, i.e., within 30 residues of the last tworesidues shown in SEQ ID NO:1), and utilize the carboxy terminalsequences therefrom.

EXAMPLE 2 Preparation of Resorter Constructs

Various reporter constructs are used in the examples which follow. Theyare prepared as follows:

TK-LUC: The MTV-LTR promoter sequence was removed from the MTV-LUCplasmid described by Hollenberg and Evans in Cell 55:899-906 (1988) byHindIII and XhoI digest, and cloned with the HindIII-XhoI fragment ofthe Herpes simplex virus thymidine kinase gene promoter (−105 to +51with respect to the transcription start site, m, isolated from plasmidpBLCAT2, described by Luckow & Schutz in Nucleic Acids Res. 15:5490(1987)) to generate parental construct TK-LUC.

pTK-PPRE3-LUC: Three copies of double-stranded peroxisome proliferatorresponse element (PPRE) oligonucleotides (see SEQ ID NO:5) were clonedupstream of the TK promoter of TK-LUC at the SalI site.

pTK-MH100×4-LUC: Four copies of double-stranded MH100 oligonucleotides,encoding a GAL4 binding site, were cloned upstream of the TK promoter ofTK-LUC at the HindIII site.

CMX-βGAL: The coding sequence for the E. coli β-galactosidase gene wasisolated from plasmid pCH110 [see Hall et al., J. Mol. Appl. Genet.2:101-109 (1983)] by HindIII and BamHI digest, and cloned into pCMXeucaryotic expression vector [see Umesono et al., supra].

EXAMPLE 3 Screening Assay for Receptor Selective Agonists

CV-1 cells are co-transfected with CMX-GAL-PPARγ and pTK-MH100×4-LUC ata ratio of about 100 ng of receptor-encoding DNA per 10⁵ cells. Theusual amounts of DNA per 10⁵ cells are 100 ng of CMX-GAL-PPARγ, 300 ngof pTK-MH100×4-LUC, and 500 ng of CMX-βGAL. Typically, transfections areperformed in triplicate. The plates are then incubated for 2-3 hours at37° C.

The cells are washed with fresh medium. Fresh medium containing oneconcentration of a serial dilution of agonist is added to each well. Atypical agonist dilution series extends from 10⁻⁵M through 10⁻¹¹M. Asolvent control is performed for each agonist. The cells are incubatedat 37° C. for 1-2 days.

The cells are rinsed twice with buffered saline solution. Subsequently,cells are lysed, in situ, by adding 200 μl of lysis buffer. After 30minutes incubation at room temperature, 40 μl aliquots of cell lysateare transferred to 96-well plates for luciferase reporter gene assaysand β-galactosidase transfection controls [see Heyman et al., Cell68:397-406 (1992)].

The data are expressed as relative light units (RLUs) per O.D. unit ofβ-galactosidase per minute. The triplicates are averaged for eachconcentration and plotted (see FIG. 1) as fold induction induced by astandard dose (10 μM) of agonist.

EXAMPLE 4 Dose Response of GAL4-PPARγ Constructs to VariousProstaglandins

Effector plasmid, reporter plasmid, and β-galactosidase control plasmidare co-transfected into CV-1 cells at a ratio of about 1:3:5, using aliposome-mediated method, employingN-{2-(2,3)-dioleoyloxy)propyl-N,N,N-trimethyl ammonium methyl sulfate}(i.e., DOTAP, Boehringer Mannheim) according to the manufacturer'sinstructions in Dulbecco's modified Eagle's medium (DMEM) with 10%delipidated hormone-depleted fetal calf serum. After about 2-3 hours,the cells are washed with DMEM and an appropriate prostaglandin is addedto the media to the final molar concentration indicated in FIG. 2. After24-48 hours of incubation, the cells are rinsed with phosphate bufferedsaline (pH 7.2) and lysed. Aliquots are assayed for luciferase andβ-galactosidase activity. Luciferase activity is normalized to opticaldensity units of β-galactosidase per minute of incubation.

The data are expressed in FIG. 2 as fold induction over the sameconstruct incubated in solvent alone. Review of FIG. 2 reveals that PGD2and PGJ2 families of compounds are functional modulators of PPARγ.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

7 1 2005 DNA Mus Musculus CDS (352)..(1776) 1 atcgaatccc gcgccccaggcgctgccgct ctgagtgcga cgggccccgc ctggccggcc 60 ggaggacgcg gaagaagagacctggggcgc tgcctggggt attgggtcgc gcgcagtgag 120 gggaccgagt gtgacgacaaggtgaccggg ctgaggggac gggctgagga gaagtcacac 180 tctgacagga gcctgtgagaccaacagcct gacggggtct cggttgaggg gacgcgggct 240 gagaagtcac gttctgacaggactgtgtga cagacaagat ttgaaagaag cggtgaacca 300 ctgatattca ggacatttttaaaaacaaga ctacccttta ctgaaattac c atg gtt 357 Met Val 1 gac aca gag atgcca ttc tgg ccc acc aac ttc gga atc agc tct gtg 405 Asp Thr Glu Met ProPhe Trp Pro Thr Asn Phe Gly Ile Ser Ser Val 5 10 15 gac ctc tcc gtg atggaa gac cac tcg cat tcc ttt gac atc aag ccc 453 Asp Leu Ser Val Met GluAsp His Ser His Ser Phe Asp Ile Lys Pro 20 25 30 ttt acc aca gtt gat ttctcc agc att tct gct cca cac tat gaa gac 501 Phe Thr Thr Val Asp Phe SerSer Ile Ser Ala Pro His Tyr Glu Asp 35 40 45 50 att cca ttc aca aga gctgac cca atg gtt gct gat tac aaa tat gac 549 Ile Pro Phe Thr Arg Ala AspPro Met Val Ala Asp Tyr Lys Tyr Asp 55 60 65 ctg aag ctc caa gaa tac caaagt gcg atc aaa gta gaa cct gca tct 597 Leu Lys Leu Gln Glu Tyr Gln SerAla Ile Lys Val Glu Pro Ala Ser 70 75 80 cca cct tat tat tct gaa aag acccag ctc tac aac agg cct cat gaa 645 Pro Pro Tyr Tyr Ser Glu Lys Thr GlnLeu Tyr Asn Arg Pro His Glu 85 90 95 gaa cct tct aac tcc ctc atg gcc attgag tgc cga gtc tgt ggg gat 693 Glu Pro Ser Asn Ser Leu Met Ala Ile GluCys Arg Val Cys Gly Asp 100 105 110 aaa gca tca ggc ttc cac tat gga gttcat gct tgt gaa gga tgc aag 741 Lys Ala Ser Gly Phe His Tyr Gly Val HisAla Cys Glu Gly Cys Lys 115 120 125 130 ggt ttt ttc cga aga acc atc cgattg aag ctt att tat gat agg tgt 789 Gly Phe Phe Arg Arg Thr Ile Arg LeuLys Leu Ile Tyr Asp Arg Cys 135 140 145 gat ctt aac tgc cgg atc cac aaaaaa agt aga aat aaa tgt cag tac 837 Asp Leu Asn Cys Arg Ile His Lys LysSer Arg Asn Lys Cys Gln Tyr 150 155 160 tgt cgg ttt cag aag tgc ctt gctgtg ggg atg tct cac aat gcc atc 885 Cys Arg Phe Gln Lys Cys Leu Ala ValGly Met Ser His Asn Ala Ile 165 170 175 agg ttt ggg cgg atg cca cag gccgag aag gag aag ctg ttg gcg gag 933 Arg Phe Gly Arg Met Pro Gln Ala GluLys Glu Lys Leu Leu Ala Glu 180 185 190 atc tcc agt gat atc gac cag ctgaac cca gag tct gct gat ctg cga 981 Ile Ser Ser Asp Ile Asp Gln Leu AsnPro Glu Ser Ala Asp Leu Arg 195 200 205 210 gcc ctg gca aag cat ttg tatgac tca tac ata aag tcc ttc ccg ctg 1029 Ala Leu Ala Lys His Leu Tyr AspSer Tyr Ile Lys Ser Phe Pro Leu 215 220 225 acc aaa gcc aag gcg agg gcgatc ttg aca gga aag aca acg gac aaa 1077 Thr Lys Ala Lys Ala Arg Ala IleLeu Thr Gly Lys Thr Thr Asp Lys 230 235 240 tca cca ttt gtc atc tac gacatg aat tcc tta atg atg gga gaa gat 1125 Ser Pro Phe Val Ile Tyr Asp MetAsn Ser Leu Met Met Gly Glu Asp 245 250 255 aaa atc aag ttc aaa cat atcacc ccc ctg cag gag cag agc aaa gag 1173 Lys Ile Lys Phe Lys His Ile ThrPro Leu Gln Glu Gln Ser Lys Glu 260 265 270 gtg gcc atc cga att ttt caaggg tgc cag ttt cga tcc gta gaa gcc 1221 Val Ala Ile Arg Ile Phe Gln GlyCys Gln Phe Arg Ser Val Glu Ala 275 280 285 290 gtg caa gag atc aca gagtat gcc aaa aat atc cct ggt ttc att aac 1269 Val Gln Glu Ile Thr Glu TyrAla Lys Asn Ile Pro Gly Phe Ile Asn 295 300 305 ctt gat ttg aat gac caagtg act ctg ctc aag tat ggt gtc cat gag 1317 Leu Asp Leu Asn Asp Gln ValThr Leu Leu Lys Tyr Gly Val His Glu 310 315 320 atc atc tac acg atg ctggcc tcc ctg atg aat aaa gat gga gtc ctc 1365 Ile Ile Tyr Thr Met Leu AlaSer Leu Met Asn Lys Asp Gly Val Leu 325 330 335 atc tca gag ggc caa ggattc atg acc agg gag ttc ctc aaa agc ctg 1413 Ile Ser Glu Gly Gln Gly PheMet Thr Arg Glu Phe Leu Lys Ser Leu 340 345 350 cgg aag ccc ttt ggt gacttt atg gag cct aag ttt gag ttt gct gtg 1461 Arg Lys Pro Phe Gly Asp PheMet Glu Pro Lys Phe Glu Phe Ala Val 355 360 365 370 aag ttc aat gca ctggaa tta gat gac agt gac ttg gct ata ttt ata 1509 Lys Phe Asn Ala Leu GluLeu Asp Asp Ser Asp Leu Ala Ile Phe Ile 375 380 385 gct gtc att att ctcagt gga gac cgc cca ggc ttg ctg aac gtg aag 1557 Ala Val Ile Ile Leu SerGly Asp Arg Pro Gly Leu Leu Asn Val Lys 390 395 400 ccc atc gag gac atccaa gac aac ctg ctg cag gcc ctg gaa ctg cag 1605 Pro Ile Glu Asp Ile GlnAsp Asn Leu Leu Gln Ala Leu Glu Leu Gln 405 410 415 ctc aag ctg aat caccca gag tcc tct cag ctg ttc gcc aag gtg ctc 1653 Leu Lys Leu Asn His ProGlu Ser Ser Gln Leu Phe Ala Lys Val Leu 420 425 430 cag aag atg aca gacctc agg cag atc gtc aca gag cac gtg cag cta 1701 Gln Lys Met Thr Asp LeuArg Gln Ile Val Thr Glu His Val Gln Leu 435 440 445 450 ctg cat gtg atcaag aag aca gag aca gac atg agc ctt cac ccc ctg 1749 Leu His Val Ile LysLys Thr Glu Thr Asp Met Ser Leu His Pro Leu 455 460 465 ctc cag gag atctac aag gac ttg tat tagcaggaaa gtcccacccg 1796 Leu Gln Glu Ile Tyr LysAsp Leu Tyr 470 475 ctgacaacgt gttccttcta ttgattgcac tattattttgagggaaaaaa atctgacacc 1856 taagaaattt actgtgaaaa agcatttaaa aacaaaaagttttagaacat gatctatttt 1916 atgcatattg tttataaaga tacatttaca atttacttttaatattaaaa attaccacat 1976 tataaaaaaa aaaaaaaaaa aggaattcc 2005 2 475PRT Mus Musculus 2 Met Val Asp Thr Glu Met Pro Phe Trp Pro Thr Asn PheGly Ile Ser 1 5 10 15 Ser Val Asp Leu Ser Val Met Glu Asp His Ser HisSer Phe Asp Ile 20 25 30 Lys Pro Phe Thr Thr Val Asp Phe Ser Ser Ile SerAla Pro His Tyr 35 40 45 Glu Asp Ile Pro Phe Thr Arg Ala Asp Pro Met ValAla Asp Tyr Lys 50 55 60 Tyr Asp Leu Lys Leu Gln Glu Tyr Gln Ser Ala IleLys Val Glu Pro 65 70 75 80 Ala Ser Pro Pro Tyr Tyr Ser Glu Lys Thr GlnLeu Tyr Asn Arg Pro 85 90 95 His Glu Glu Pro Ser Asn Ser Leu Met Ala IleGlu Cys Arg Val Cys 100 105 110 Gly Asp Lys Ala Ser Gly Phe His Tyr GlyVal His Ala Cys Glu Gly 115 120 125 Cys Lys Gly Phe Phe Arg Arg Thr IleArg Leu Lys Leu Ile Tyr Asp 130 135 140 Arg Cys Asp Leu Asn Cys Arg IleHis Lys Lys Ser Arg Asn Lys Cys 145 150 155 160 Gln Tyr Cys Arg Phe GlnLys Cys Leu Ala Val Gly Met Ser His Asn 165 170 175 Ala Ile Arg Phe GlyArg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu 180 185 190 Ala Glu Ile SerSer Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp 195 200 205 Leu Arg AlaLeu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe 210 215 220 Pro LeuThr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr 225 230 235 240Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly 245 250255 Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser 260265 270 Lys Glu Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val275 280 285 Glu Ala Val Gln Glu Ile Thr Glu Tyr Ala Lys Asn Ile Pro GlyPhe 290 295 300 Ile Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys TyrGly Val 305 310 315 320 His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu MetAsn Lys Asp Gly 325 330 335 Val Leu Ile Ser Glu Gly Gln Gly Phe Met ThrArg Glu Phe Leu Lys 340 345 350 Ser Leu Arg Lys Pro Phe Gly Asp Phe MetGlu Pro Lys Phe Glu Phe 355 360 365 Ala Val Lys Phe Asn Ala Leu Glu LeuAsp Asp Ser Asp Leu Ala Ile 370 375 380 Phe Ile Ala Val Ile Ile Leu SerGly Asp Arg Pro Gly Leu Leu Asn 385 390 395 400 Val Lys Pro Ile Glu AspIle Gln Asp Asn Leu Leu Gln Ala Leu Glu 405 410 415 Leu Gln Leu Lys LeuAsn His Pro Glu Ser Ser Gln Leu Phe Ala Lys 420 425 430 Val Leu Gln LysMet Thr Asp Leu Arg Gln Ile Val Thr Glu His Val 435 440 445 Gln Leu LeuHis Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His 450 455 460 Pro LeuLeu Gln Glu Ile Tyr Lys Asp Leu Tyr 465 470 475 3 546 DNA Saccharomycescerevisiae CDS (35)..(544) 3 gggagaccca agcttgaagc aagcctcctg aaag atgaag cta ctg tct tct atc 55 Met Lys Leu Leu Ser Ser Ile 1 5 gaa caa gcatgc gat att tgc cga ctt aaa aag ctc aag tgc tcc aaa 103 Glu Gln Ala CysAsp Ile Cys Arg Leu Lys Lys Leu Lys Cys Ser Lys 10 15 20 gaa aaa ccg aagtgc gcc aag tgt ctg aag aac aac tgg gag tgt cgc 151 Glu Lys Pro Lys CysAla Lys Cys Leu Lys Asn Asn Trp Glu Cys Arg 25 30 35 tac tct ccc aaa accaaa agg tct ccg ctg act agg gca cat ctg aca 199 Tyr Ser Pro Lys Thr LysArg Ser Pro Leu Thr Arg Ala His Leu Thr 40 45 50 55 gaa gtg gaa tca aggcta gaa aga ctg gaa cag cta ttt cta ctg att 247 Glu Val Glu Ser Arg LeuGlu Arg Leu Glu Gln Leu Phe Leu Leu Ile 60 65 70 ttt cct cga gaa gac cttgac atg att ttg aaa atg gat tct tta cag 295 Phe Pro Arg Glu Asp Leu AspMet Ile Leu Lys Met Asp Ser Leu Gln 75 80 85 gat ata aaa gca ttg tta acagga tta ttt gta caa gat aat gtg aat 343 Asp Ile Lys Ala Leu Leu Thr GlyLeu Phe Val Gln Asp Asn Val Asn 90 95 100 aaa gat gcc gtc aca gat agattg gct tca gtg gag act gat atg cct 391 Lys Asp Ala Val Thr Asp Arg LeuAla Ser Val Glu Thr Asp Met Pro 105 110 115 cta aca ttg aga cag cat agaata agt gcg aca tca tca tcg gaa gag 439 Leu Thr Leu Arg Gln His Arg IleSer Ala Thr Ser Ser Ser Glu Glu 120 125 130 135 agt agt aac aaa ggt caaaga cag ttg act gta tcg ccg gaa ttc ccg 487 Ser Ser Asn Lys Gly Gln ArgGln Leu Thr Val Ser Pro Glu Phe Pro 140 145 150 ggg atc cgt cga cgg taccag ata tca gga tcc tgg cca gct agc tag 535 Gly Ile Arg Arg Arg Tyr GlnIle Ser Gly Ser Trp Pro Ala Ser 155 160 165 gta gct aga gg 546 Val AlaArg 4 166 PRT Saccharomyces cerevisiae 4 Met Lys Leu Leu Ser Ser Ile GluGln Ala Cys Asp Ile Cys Arg Leu 1 5 10 15 Lys Lys Leu Lys Cys Ser LysGlu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 Lys Asn Asn Trp Glu Cys ArgTyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45 Leu Thr Arg Ala His Leu ThrGlu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60 Glu Gln Leu Phe Leu Leu IlePhe Pro Arg Glu Asp Leu Asp Met Ile 65 70 75 80 Leu Lys Met Asp Ser LeuGln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95 Phe Val Gln Asp Asn ValAsn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110 Ser Val Glu Thr AspMet Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115 120 125 Ala Thr Ser SerSer Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140 Thr Val SerPro Glu Phe Pro Gly Ile Arg Arg Arg Tyr Gln Ile Ser 145 150 155 160 GlySer Trp Pro Ala Ser 165 5 13 DNA Mus musculus 5 aggacaaagg tca 13 6 23DNA Mus musculus 6 ggaccaggac aaaggtcacg ttc 23 7 17 DNA Saccharomycescerevisiae 7 cggaggactg tcctccg 17

That which is claimed is:
 1. A method of testing a compound for itsability to regulate transcription-activating effects of a peroxisomeproliferator activated receptor-gamma (PPAR-γ), said method comprisingassaying for changes in the level of reporter protein preset as a restof contacting cells containing said receptor and reporter vector withsaid compound; wherein said receptor is introduced into said cells by areceptor expression vector comprising a DNA segment encoding PPAR-γ, andwherein said reporter vector comprises: (a) a promoter that is operablein said cell, (b) a hormone response element; wherein said hormoneresponse element is a direct repeat of two or more half sites separatedby a spacer of one nucleotide, wherein said spacer can be A, C, G or T,wherein each half site comprises the sequence -RGBNNM-,  wherein R isselected from A or G; B is selected form G, C, or T; each N isindependently selected from A, T, C, or G; and M is selected from A orC;  with the proviso that at least 4 nucleotides of said-RGBNNM-sequence are identical with the nucleotides at correspondingpositions of the sequence -AGGTCA-; and wherein said response element isoptionally preceded by N_(x), wherein x falls in the range of 0 up to 5,and (c) a DNA segment encoding a reporter protein, wherein said reporterprotein-encoding DNA segment is operatively linked to said promoter fortranscription of said DNA segment, and wherein said hormone responseelement is operatively linked to said promoter for activation thereof,wherein an increase or decrease in the level of the reporter proteinwhen said cells are contacted with said compound, relative to the levelof the reporter protein when said cells are not contacted with saidcompound, is indicative of a compound that regulates thetranscription-activating effects of said receptor.
 2. A method accordingto claim 1 wherein said response element has at least one copy of theminimal sequence: AGGACA A AGGTCA (SEQ. ID NO. 5), wherein said minimalsequence is optionally flanked by additional residues.
 3. A methodaccording to claim 1 wherein said response element has at least one copyof the sequence: GGACC AGGACA A AGGTCA CGTTC (SEQ. ID NO. 6).
 4. Amethod of testing a compound for its ability to regulatetranscription-activating effects of a peroxisome proliferator activatedreceptor-gamma (PPAR-γ), said method comprising assaying for changes inthe level of reporter protein preset as a rest of contacting cellscontaining said receptor and reporter vector with said compound; whereinsaid receptor is introduced into said cells by a receptor expressionvector comprising a DNA segment encoding PPAR-γ, and wherein saidreporter vector comprises: (a) a promoter that is operable in said cell,(b) a hormone response element; (c) a DNA segment encoding a reporterprotein, wherein said reporter protein-encoding DNA segment isoperatively linked to said promoter for transcription of said DNAsegment, and wherein said hormone response element is operatively linkedto said promoter for activation thereof, wherein said compound is aputative antagonist for said PPAR-γ, and wherein said contacting iscarried out in the presence of increasing concentrations of saidcompound, and a fixed concentration of at low one agonist for saidPPAR-γ, wherein a decrease in the level of the reporter protein whensaid cells are contacted with said compound and said agonist, relativeto the level of there reporter protein when said cells are contactedwith said agonist alone, is indicative of a compound that is anantagonist of said receptor.
 5. A method of testing a compound for itsability to regulate transcription-activating effects of a peroxisomeproliferator activated receptor-gamma (PPAR-γ), said method comprisingassaying for changes in the level of reporter protein preset as a restof contacting cells containing said receptor and reporter vector with(i) a test compound, and (ii) at least compound, that is a PPAR-γantagonist; wherein said receptor is introduced into said cells by areceptor expression vector comprising a DNA segment encoding PPAR-γ, andwherein said reporter vector comprises: (a) a promoter that is operablein said cell, (b) a hormone response element; (c) a DNA segment encodinga reporter protein, wherein said reporter protein-encoding DNA segmentis operatively linked to said promoter for transcription of said DNAsegment, and wherein said hormone response element is operatively linkedto said promoter for activation thereof, wherein an increase or decreasein the level of the reporter protein when said cells are contacted withsaid compound, relative to the level of the reporter protein when saidcells are not contacted with said compound, is indicative of a compoundthat regulates the transcription-activating effects of said receptor. 6.A method of testing a compound for its ability to regulatetranscription-activating effects of a peroxisome proliferator activatedreceptor-gamma (PPAR-γ), said method comprising assaying for changes inthe level of reporter protein preset as a rest of contacting cellscontaining a GAL4 chimeric PPAR-γ receptor and a reporter vector with atest compound; wherein said GAL4 chimeric PPAR-γ receptor is introducedinto said cells by a receptor expression vector comprising a DNA segmentencoding at least the ligand binding domain of a PPAR-γ and a DNAsegment encoding a GAL4 DNA binding domain, wherein the DNA segmentencoding said GAL4 DNA binding domain is introduced at the carboxyterminus of the DNA segment encoding said ligand binding domain of aPPAR-γ, and wherein said reporter vector comprises: (a) a promoter thatis operable in said cell, (b) a GAL4 response element capable of beingbound by said GAL4 DNA binding domain, and (c) a DNA segment encoding areporter protein, wherein said reporter protein-encoding DNA segment isoperatively linked to said promoter for transcription of said DNAsegment, and wherein said GAL4 response element is operatively linked tosaid promoter for activation thereof, wherein an increase or decrease inthe level of the reporter protein when said cells are contacted withsaid compound, relative to the level of the reporter protein when saidcells are not contacted with said compound, is indicative of a compoundthat regulates the transcription-activating effects of said receptor. 7.A method according to claim 6, wherein the DNA segment encoding saidGAL4 DNA binding domain encodes amino acid residues 1-147 of the GAL4protein.
 8. A method according to claim 6, wherein the DNA segmentencoding said GAL4 DNA binding domain encodes amino acid residues 1-90of the GAL4 protein.
 9. A method according to claim 6, wherein the DNAsegment encoding said GAL4 DNA binding domain encodes amino acidresidues 1-74 of the GAL4 protein.
 10. A method of testing a compoundfor its ability to regulate transcription-activating effects of aperoxisome proliferator activated receptor-gamma (PPAR-γ), said methodcomprising assaying for changes in the level of reporter protein presetas a rest of contacting cells containing a GAL4 chimeric PPAR-γ receptorand a reporter vector with said compound; wherein said GAL4 chimericPPAR-γ receptor is introduced into said cells by a receptor expressionvector comprising a DNA segment encoding at least the ligand bindingdomain of a PPAR-γ and a DNA segment encoding a GAL4 DNA binding domain,and wherein said reporter vector comprises: (a) a promoter that isoperable in said cell, (b) a GAL4 response element capable of beingbound by said GAL4 DNA binding domain, and (c) a DNA segment encoding areporter protein, wherein said reporter protein-encoding DNA segment isoperatively linked to said promoter for transcription of said DNAsegment, and wherein said hormone response element is operatively linkedto said promoter for activation thereof, wherein said compound is aputative antagonist for said PPAR-γ, and wherein said contacting iscarried out in the presence of increasing concentrations of saidcompound, and a fixed concentration of at low one agonist for saidPPAR-γ, wherein a decrease in the level of the reporter protein whensaid cells are contacted with said compound and said agonist, relativeto the level of there reporter protein when said cells are contactedwith said agonist alone, is indicative of a compound that is anantagonist of said receptor.
 11. A method of testing a compound for itsability to regulate transcription-activating effects of a peroxisomeproliferator activated receptor-gamma (PPAR-γ), said method comprisingassaying for changes in the level of reporter protein preset as a restof contacting cells containing a GAL4 chimeric PPAR-γ receptor and areporter vector with (i) a test compound, and (ii) at least oneadditional compound that is a PPAR-γ agonist; wherein said GAL4 chimericPPAR-γ receptor is introduced into said cells by a receptor expressionvector comprising a DNA segment encoding at least the ligand bindingdomain of a PPAR-γ and a DNA segment encoding a GAL4 DNA binding domain,wherein the DNA segment encoding said GAL4 DNA binding domain isintroduced at the carboxy terminus of the DNA segment encoding saidligand binding domain of a PPAR-γ, and wherein said reporter vectorcomprises: (a) a promoter that is operable in said cell, (b) a GAL4response element capable of being bound by said GAL4 DNA binding domain,and (c) a DNA segment encoding a reporter protein, wherein said reporterprotein-encoding DNA segment is operatively linked to said promoter fortranscription of said DNA segment, and wherein said GAL4 responseelement is operatively linked to said promoter for activation thereof,wherein an increase or decrease in the level of the reporter proteinwhen said cells are contacted with said compound, relative to the levelof the reporter protein when said cells are not contacted with saidcompound, is indicative of a compound that regulates thetranscription-activating effects of said receptor.
 12. A method oftesting a compound for its ability to regulate transcription-activatingeffects of a peroxisome proliferator activated receptor-gamma (PPAR-γ),said method comprising assaying for changes in the level of reporterprotein preset as a rest of contacting cells containing said receptorand reporter vector with (i) a test compound, and (ii) at leastcompound, that is a PPAR-γ antagonist; wherein said GAL4 chimeric PPAR-γreceptor is introduced into said cells by a receptor expression vectorcomprising a DNA segment encoding at least the ligand binding domain ofa PPAR-γ and a DNA segment encoding a GAL4 DNA binding domain, andwherein said reporter vector comprises: (a) a promoter that is operablein said cell, (b) a GAL4 response element capable of being bound by saidGAL4 DNA binding domain, and (c) a DNA segment encoding a reporterprotein, wherein said reporter protein-encoding DNA segment isoperatively linked to said promoter for transcription of said DNAsegment, and wherein said GAL4 response element is operatively linked tosaid promoter for activation thereof, wherein an increase or decrease inthe level of the reporter protein when said cells are contacted withsaid compound, relative to the level of the reporter protein when saidcells are not contacted with said compound, is indicative of a compoundthat regulates the transcription-activating effects of said receptor.